Certain strains of Escherichia coli are capable of causing a diarrheal disease in man which resembles Shigellosis. Pathogenesis of the disease involves invasion of colonic epithelial cells, intracellular multiplication and tissue destruction. Enteroinvasive Escherichia coli contain a large plasmid required for epithelial cell invasion, and intracellular multiplication. This plasmid shares considerable DNA homology with the virulence plasmid of S. flexneri. The goals of this project are 1) to identify plasmid encoded- determinants required for entry into and multiplication within epithelial cells 2) to identify plasmid-encoded gene products required for virulence. The definition of plasmid-encoded virulence determinants will be accomplished by a combination of cloning and transposon mutagenesis methods. The expression of cloned virulence determinants will be studied using minicells and an E. coli in vitro transcription translation system. The ability to enter and survive within intestinal epithelial cells is a trait shared by a large number of enteric pathogens including enteroinvasive Escherichia coli, Shigella, Salmonella and Yersinia enterocolitica. Despite this, very little is known about how a bacterium gains entry into its host cell. One reasonable hypothesis is that invasive bacteria have proteins on their surface which react with host-cell receptors to trigger their own uptake-- -that is, that they enter the host cell via receptor-mediated endocytosis. The identification of invasion determinants in enteroinvasive E. coli will lead to a better understanding of this entry process both in E. coli and in Shigella. This work should also facilitate research on events in pathogenesis subsequent to invasion such as intracellular survival and multiplication.